PROJECT SUMMARY More than 15,000 patients receive life-saving kidney transplants in the US every year. Nevertheless, frequent post-transplant complications limit the outlook for kidney transplant recipients. Urinary tract infections (UTIs), including multi-drug antibiotic-resistant infections, occur at an alarmingly high rate after kidney transplantation. At least 20% of kidney transplant recipients experience a serious UTI in the first 3 months after transplantation. UTI has been associated with development of urosepsis, decreasing graft function, graft loss, and death. The current gold standard for diagnosis of UTI is bacterial culture, which is fundamentally limited by its ability to detect only culturable bacteria. Furthermore, bacterial culture does not inform about co-infections, commensal microbiota, or the host response to infection. New biochemical and genetic approaches developed by our groups can fundamentally change the paradigm for UTI diagnosis and transplant monitoring. The goal of this proposal is to develop and apply precision medicine approaches for the monitoring of UTIs after kidney transplantation. We will utilize (1) newly developed technologies for the metagenomic sequencing of ultrashort fragments of cfDNA in urine, (2) new metagenomic host-response measurements, (3) technologies to perform bioinformatic sorting of resistance determinants, and (4) new approaches to perform rapid cfDNA metagenomic sequencing and direct profiling of epigenetic markers comprised within cfDNA based on Oxford Nanopore Technologies. In the first Aim, we will investigate the utility of metagenomic sequencing of urinary cfDNA as a tool to monitor UTIs and to profile the urinary microbiome. This concept is supported by extensive pilot data. In the second Aim we will investigate a cfDNA metagenomic sequencing assay that informs the degree of host injury due to infection. This second Aim is also supported by significant results from pilot data. In the third Aim, we will investigate the performance of novel, Nanopore sequencing technologies and algorithms that will enable a lower cost, faster turnaround time, and greater utility of cfDNA metagenomic sequencing assays. Together, these assays will provide new avenues to monitor infections of the urinary tract, profile antibiotic resistance, describe the urinary microbiome, and examine host-pathogen interactions. More than 15,000 patients receive kidney transplants each year, and many of these patients suffer complications from UTI. In the general population, UTI is one of the most common medical problems, with 150 million persons affected per year worldwide. Successful implementation of these studies will provide new avenues for the monitoring of bacterial UTI and will thereby address an acute medical need in kidney transplantation, and by extension, a substantial health benefit to the general public.